Many factors affect gene expression in plants and other eukaryotic organisms. Recently, small RNAs, 21-26 nucleotides, have emerged as important regulators of eukaryotic gene expression. The known small regulatory RNAs fall into two basic classes. One class of small RNAs is the short interfering RNAs (siRNAs). These play essential roles in RNA silencing, a sequence-specific RNA degradation process that is triggered by double-stranded RNA (dsRNA) (see Vance and Vaucheret (2001) Science 292:2277-2280, and Zamore (2001) Nat Struct Biol 8:746-750 for recent reviews on RNA silencing in plants and animals, respectively). RNA silencing plays a natural role in defense against foreign nucleic acids including virus resistance in plants and control of transposons in a number of organisms. siRNAs are double-stranded with small 3′ overhangs and derive from longer dsRNAs that induce silencing. They serve as guides to direct destruction of target RNAs and have been implicated as primers in the amplification of dsRNA via the activity of a cellular RNA dependent RNA polymerase. In plants, si-like RNAs have also been associated with dsRNA-induced transcriptional gene silencing (TGS), a process in which dsRNA with homology to promoter regions triggers DNA methylation and inhibits transcription. The TGS-associated small RNAs, unlike true siRNAs, are not involved in RNA degradation.
Another group of small RNAs are known generically as short temporal RNAs (stRNAs) and more broadly as micro-RNAs (miRNAs). miRNAs have emerged as evolutionarily conserved, RNA-based regulators of gene expression in animals and plants. miRNAs (approx. 21 to 25 nt) arise from larger precursors with a stem loop structure that are transcribed from non-protein-coding genes. miRNAs are single-stranded, and their accumulation is developmentally regulated and/or regulated by environmental stimuli. They derive from partially double-stranded precursor RNAs that are transcribed from genes that do not encode protein. The miRNAs appear to be transcribed as hairpin RNA precursors, which are processed to their mature, about 21 nt forms by Dicer (Lee R D, and Ambros, V. Science 294: 862-864 (2001)). miRNA targets a specific mRNA to suppress gene expression at post-transcriptional level (i.e. degrades mRNA) or at translational level (i.e. inhibits protein synthesis). microRNAs (miRNAs) have emerged as evolutionarily conserved. There are several hundred of miRNAs have been recently identified through computational analysis and experimental approaches from many plant and animal species. A body of miRNAs is well conserved within plant kingdom or animal kingdom, but some are species or genus specific.
miRNA genes are first transcribed by Pol II RNA polymerase resulting in pri-miRNA with Cap structure at 5′ end and poly tail at 3′ end. Pri-miRNA is subjected to cleavage by an RNase III-like enzyme, Dicer, to generate mature miRNA. miRNA is then recruited into RISC (RNA induced silencing complex) and targets a specific mRNA in cytoplasm to suppress gene expression at post-transcriptional level (i.e. degrades mRNA). MiRNA can also inhibit protein synthesis after targeting a mRNA in a sequence-specific manner. The mechanism of such translational inhibition is to be uncovered. It has been shown both in animal and plant, pairing of the miRNA 5′ region to its target mRNA is crucial for miRNA actions (Mallory A et al., EMBO Journal 23:3356-3364, 2004; Doench J and Sharp P, Genes & Development 504-511, (2004)).
Thus, it was realized that small, endogenously encoded hairpin RNAs could stably regulate gene expression via elements of the RNAi machinery. Like stRNAs (and unlike siRNAs involved in RNA silencing), most of the miRNAs lack complete complementarity to any putative target mRNA. Although their functions are, as yet, not known, it is hypothesized that they regulate gene expression during development, perhaps at the level of development. However, given the vast numbers of these small regulatory RNAs, it is likely that they are functionally more diverse and regulate gene expression at more than one level. In plant, majority of miRNA target genes are transcription factors which are required for meristem identity, cell division, organ separation, and organ polarity. Some miRNAs have unique tissues-specific and/or temporal expression pattern. McManus et al. (RNA 8:842-850 (2002)) also studied miRNA mimics containing 19 nucleotides of uninterrupted RNA duplex, a 12-nucleotide loop length and one asymmetric stem-loop bulge composed of a single uridine opposing a double uridine. Synthetic miRNA can either be transfected into cells or expressed in the cell under the control of an RNA polymerase III promoter and cause the decreased expression of a specific target nucleotide sequence (McManus et al. (2002) RNA 8:842-850, herein incorporated by reference).
In plant, there have been increasing evidences that microRNAs target genes involved in many aspects of plant growth and development such as meristem identity, cell division, organ separation, and organ polarity. For example, miR164 targets NAC-domai genes, which encodes a family of transcription factors including (CUP-SHAPED COTYLEDON1, CUC1 and CUC2). Expression of miR164-resistant version of CUC1 mRNA from the CUC1 promoter causes alterations in Arabidopsis embryonic vegetative, and floral development (Mallory A et al., Current Biology 14:1035-1046, (2004)). MiR166 mediates leaf polarity in Arabidopsis and maize (Juarez M et al., Nature 428: 84-88, (2004) and Kidner C and Martlenssen R, Nature 428: 81-84, (2004)). MiR172 directs flower development through regulating APETALA2 gene expression (Chen X, Science, 303: 2022-2025 (2004)). MiRNAs also regulate plant gene expression in response to environmental stimuli such as abiotic stress. For example, the expression of miR395, the sulfurylase-targeting miRNA, is increased upon sulfate starvation (Jone-Rhoades M W and Bartel D, Molecular Cell 14: 787-799, (2004)). MiR319c expression is upregulated by cold but not dehydration, NaCl or ABA (Sunkar R and Zhu J K, The Plant Cell 16:2001:2019, (2004)). Some miRNAs have unique tissues-specific and/or temporal expression patterns. For example, miR398b is expressed predominantly in Arabidopsis leaf (Sunkar R and Zhu J K., The Plant Cell 16:2001:2019, 2004)
In animals, miRNAs also play a key role in growth and development. For example, in mammals, miR181 modulates hematopietic lineage differentiation (Chen C Z et al., Science 303:83-86, (2004)), and MiR196 direct cleavage of HOXB8 mRNA (Yekta S et al., Science 304:594-596, (2004)). In human, miR-124 is expressed only in brain with possible role in neuronal differentiation (Sempere L. F. et al., Genome Biology 5:R13 (2004)) while miR-1 is expressed in muscle (Lagos-Quintana. M et al., Current Biology, (2002))
In plant, so far disclosed applications of miRNAs are    1) overexpression and/or ectopic expression of a given miRNA to characterize its function or generate desired phenotypes (Palatnik J et al., Nature 425: 257-263, (2003));    2) engineering a miRNA precursor to produce new miRNA targeting gene-of-interest (WO2004009779;    3) engineering mRNA to be resistant to miRNA recognition and cleavage (i.e. silent mutation—by changing nucleotides in the codons for the same amino acid) (Palatnik J et al., Nature 425: 257-263, 2003; Mallory A et al., Current Biology 14:1035-1046, (2004)).
US 20040268441 describes microRNA precursor constructs that can be designed to modulate expression of any nucleotide sequence of interest, either an endogenous plant gene or alternatively a transgene.
One of the major obstacles in various field of biotechnology (including but not limited to gene therapy and plant biotechnology) is the difficulty to achieve cell or tissue specificity. Transcription is an essential process for every living organism to convert abstract genetic information into physical reality. Promoter is a major component to drive transcription. Some promoters are active in every tissue (e.g. actin promoters) while other promoters only active in limited tissues. It is quite often that a given promoter is predominantly active in one tissue type but weakly expressed in some other tissues, so called leaky promoters. Those promoters are undesirable for agriculture and pharmaceutical application because unintended expression of gene-of-interest resulted from leaky promoters could cause detrimental effects to crops or patients. It certainly would not meet requirement of regulatory agency.
For example plant-parasitic nematodes cause diseases in all crops of economic importance, resulting in an estimated US$100 billion annual losses to world agriculture. In US, soybean cyst nematode is No. 1 pest—infecting nearly all soybean production states (approx. 80 million acres) and causes up to 30% yield loss each year. Chemical control measures are inadequate and environmentally unfriendly. Transgenic-plant technology offers a great potential, however, no significant success has been made yet. One major problem is the leaky activities of nematode feeding site ‘specific’ promoter. Although such promoter (e.g. TobRB7) could drive phytotoxic molecules to ‘kill’ the feeding cells and alleviate nematode infection, leaky expression of these phytotoxic molecules in other tissues (e.g. flower) causes detrimental effects on the host plants. Thus, a novel approach to control leaky expression is in high demand.
For example a major problem in chemotherapy and radiation therapy for cancer is the difficulty in achieving tumor-specific cell killing. The inability of radiation or cytotoxic chemotherapeutic agents to distinguish between tumor cells and normal cells necessarily limits the dosage that can be applied. As a result, disease relapse due to residual surviving tumor cells is frequently observed, and thus there exists a clear need for alternative non-surgical strategies. Development of gene therapy techniques is approaching clinical realization for the treatment of neoplastic and metabolic diseases, and numerous genes displaying anti-tumor activity have been identified. However, the usefulness of gene therapy methods has been limited due to systemic toxicity of anti-tumor polypeptides encoded by gene therapy constructs (Spriggs & Yates (1992) in Bentler, ed., Tumor Necrosis Factor: The Molecules and Their Emerging Roles in Medicine, pp. 383-406 Raven Press, New York, N.Y.; Sigel & Puri (1991) J Clin Oncol 9:694-704; Ryffel (1997) Immunopathol 83:18-20). Problems with current state-of-the-art gene therapy strategies include the inability to deliver the therapeutic gene specifically to the target cells. This leads to toxicity in cells that are not the intended targets. For example, manipulation of-the p53 gene suppresses the growth of both tumor cells and normal cells, and intravenous administration of tumor necrosis factor alpha (TNF.alpha.) induces systemic toxicity with such clinical manifestations as fever and hypertension. Attempts have been made to overcome these problems. These include such strategies as the use of tissue-specific promoters to limit gene expression to specific tissues and the use of heat (Voellmy R., et al., Proc. Natl. Acad. Sci. USA, 82:4949-4953 (1985)) or ionizing radiation inducible enhancers and promoters (Trainman, R. H., et al., Cell 46: 567-574 (1986); Prowess, R., et al., Proc. Natl. Acad. Sci. USA 85, 7206-7210 (1988)) to enhance expression of the therapeutic gene in a temporally and spatially controlled manner.
Adenoviral vectors possess a number of attributes that render them useful gene delivery vehicles for systemic gene therapy. Ideally, such a system would be designed so that systemically administered vector would home specifically to tumor target cells without ectopic infection of normal cells. However, a major stumbling block to this approach is the fact that the majority of adenoviral vectors administered systemically are sequestered in the liver. Therefore measures that specifically control the distribution of delivered transgene expression must be superimposed on the basic vector for optimal applicability of adenoviral vectors.
Unfortunately, for most of the presently expression systems expression of the active ingrediant is not restricted to the tumor sites due to the ‘leakiness’ of the available promoters thereby limiting efficiency of such approaches. Tissue specific promoters may add a further degree of transgene expression selectivity but there are few of these that have been validated in vivo and all are subject to some degree of non-specific activation or “leakiness”. A versatile mechanism for controllable gene expression is therefore highly desired for gene therapy.
A mechanism for controlling gene expression should ideally include both spatial and temporal control of gene expression. One existing strategy employs a chemically regulated signal, for example the tetracycline-inducible gene expression system (Gossen & Bujard (1992) Proc Natl Acad Sci USA 89:5547-5551; Gossen & Bujard (1993) Nuc Acids Res 21(18):4411-4412; Gossen et al. (1995) Science 268:1766-1769). A similar approach involves the provision of ionizing radiation to activate a radiosensitive promoter, e.g. the EGR-1 promoter (Weischelbaum et al. (1994) Cancer Res 54:4266-4269; Hallahan et al. (1995) Nat Med 1(8):786-791; Joki et al. (1995) Hum Gen Ther 6:1507-1513). An alternative design relies on endogenous control of gene expression. For example, the CEA promoter is selectively expressed in cancer cells (Hauck & Stanners (1995) J Biol Chem 270:3602; Richards et al. (1995) Human Gene Ther 6:881-893).
In the past, several approaches have been attempted to solve leakiness problem in plant gene expression without much success. By conducting a series of deletion of promoter sequence, one might eliminate the sequence in the promoter region which contributes to the leaky expression. For example, a deleted version of TbRB7 promoter drives GUS reporter gene expression in nematode feeding cells in the root upon nematode infection. Leaky expression, however, in flower tissue is still unsolved (Opperman C H et al., Science 263:221-223, (1994)). By making a chimeric promoter, i.e. a minimal promoter (e.g. 35S promoter) plus tissues-specific regulatory elements, one might restrict gene expression in desired tissues. However, if tissue-specific regulatory elements are leaky, the chimeric promoter will be leaky as well.
US 20030045495 is disclosing modified inducible systems for selective expression of therapeutic genes by hyperthermia. However, hypothermia is also difficult to be applied to discrete cells or small tissue areas.
US 20010049828 is describing a method and system for controlling the expression of transgene products in specific tissues in a transgenic animal. The system is based on an interaction of various transactivators. The transcactivator activity is controlled by antisense which is under control of tissue-specific promoters, thereby suppressing expression in certain tissues. The system is rather complicated and relies on serveral expression constructs and transgenic transcription factors. A similar system is described in US 20020065243.
US 20020022018 described control of tissue-specificity by employing tissue-specific deletion or destruction of the expression-construct in the target organism by tissue-specific expression of a Cre recombinase. As a result of Cre recombinase expression, the same or another vector that expresses the transgene in that tissue will be cut by the action of the Cre recombinase into several pieces due to LoxP sites that are strategically placed within the vector backbone. Consequently, unwanted transgene as well as viral gene expression are prevented. However, due to leakiness of the promoter driving Cre expression, expression is expected to be lowered also in the target tissue itself, thereby decreasing overall efficiency of this approach.
Although each of the afore-mentioned systems display inducibility thereby solving problem with the temporal control of gene expression, the spatial precision of gene induction is still lacking. All systems disclosed in the art so far are either highly complex and/or also reducing efficient expression in the target cells. Thus, there remains substantial need for improvement of tissue-specificity or control of promoter leakiness. The present invention provides such means and methods thereby fulfilling this longstanding need and desire in the art.